KR102089938B1 - Use of extracellular vesicles from kefir grain - Google Patents

Abstract

The present invention relates to various uses using extracellular vesicles derived from kefir grains, and more specifically, effectively preventing inflammatory diseases, bowel disease and mental disorders using kefir grain derived microvesicles. , It relates to a composition that can be improved or treated.

Description

Use of extracellular vesicles from kefir grain

The present invention relates to various uses using extracellular vesicles derived from kefir grains, and more specifically, effectively preventing inflammatory diseases, bowel disease and mental disorders using kefir grain derived microvesicles. , It relates to a composition that can be improved or treated.

Inflammation is the manifestation of a normal and protective in vivo defense mechanism that appears locally against physical trauma, harmful chemicals, microbial infections or tissue damage caused by irritating substances in metabolites in vivo. Such inflammation is triggered by various chemical mediators produced from damaged tissue and migrating cells, and these chemical mediators are known to vary according to the type of inflammatory process. In the normal case, the living body neutralizes or eliminates the onset of the disease through an inflammatory reaction and regenerates the upper tissue to restore normal structure and function, but in other cases, it progresses to a disease state such as chronic inflammation. In addition, if irritation is improperly triggered by an autoimmune reaction such as asthma, rheumatoid arthritis, or a harmless substance such as pollen, the defense reaction itself damages tissues, and thus a inflammatory disease prevention or treatment agent is needed. Inflammatory responses can be observed in almost all clinical diseases, and among these inflammatory diseases, there are some bacterial diseases that can be causally treated with antibiotics, but most have no specific treatment because the onset is due to tissue damage caused by autoimmune reactions. It is known as incurable disease.

The most common inflammatory disease prevention or treatment agents for treating these inflammatory diseases are largely divided into steroidal and nonsteroidal inflammatory disease prevention or treatment agents, and most of the synthetic inflammatory disease prevention or treatment agents have various side effects in addition to their main function. There are many disadvantages that accompany. That is, in order to treat inflammatory diseases, appropriate nonsteroidal inflammatory disease prevention or treatment drugs (NSAIDs) are used to alleviate inflammation, and if the inflammation is severe or the efficacy of NSAIDs is not effective, steroid preparations are used. I will perform surgical therapy. The NSAIDs used in this case mainly suppress cyclooxygenase (COX), thereby inhibiting the production of prostaglandin, which is involved in the inflammatory reaction, and thus exhibits the effect of preventing or treating inflammatory diseases, but gastrointestinal disorders, liver disorders, There is a problem that long-term use is difficult due to side effects such as renal dysfunction.

On the other hand, irritable bowel syndrome (IBS) is a common bowel dysfunction, which is usually characterized by persistent or repetitive abdominal pain, abdominal discomfort, and changed bowel habits. Irritable bowel syndrome impairs the patient's social and personal life, reducing the quality of life, and the more severe the symptoms, the worse the quality of life.

The causes of irritable bowel syndrome have not been clearly identified, but stress, sensitive personality, irregular eating habits, chronic fatigue, and irritating and fatty foods are known as the main causes. However, the effectiveness of the treatment methods presented so far is not large, and there is no known fundamental treatment method for the present disease, and relies solely on symptomatic treatment methods focused on symptom relief.

Recently, the number of patients with irritable bowel syndrome increased to about 1.62% in 2012, an increase of about 8.7% (130,000) over the past five years, increasing by an average of 1.7% per year. Most patients (99.4% of all patients) visited and were symptomatic Treatment with therapy. Therefore, it is urgent to develop new food biomaterials that can activate intestinal functions in addition to symptomatic treatments.

One object of the present invention is to provide a composition capable of effectively preventing, improving or treating inflammatory diseases using extracellular vesicles derived from Kefir grain.

Another object of the present invention is to provide a composition capable of effectively preventing, improving or treating intestinal diseases using kefir grain-derived microvesicles.

Another object of the present invention is to provide a composition capable of effectively preventing, improving or treating mental disorders using kefir grain-derived microvesicles.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one embodiment of the invention, kefir grains (Kefir grain) derived from microvesicles (extracellular vesicles) relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including as an active ingredient.

In the present invention, the "kefir (kefir)" is a kind of fermented milk derived from the Caucasus mountainous region, Tibetan local monks have been drinking for health is a popular drink. Kefir's constituent nutrients include proteins and polysaccharides, and vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin D, vitamin K2, folic acid, nicotinic acid and calcium, iron, and iodine. Such kefir is a yogurt-like beverage obtained by fermenting milk with a bacterial species called kefir grain (also called kefir granules), which is a complex of lactic acid bacteria and / or yeast.

In the present invention, the "kefir grain (kefir grain)" is a fermentation source used in the manufacturing process of kefir, essentially containing lactic acid bacteria, may further include yeast. Specifically, in the present invention, the lactic acid bacteria belonging to the kefir grain include Lactobacillus genus, Lactococcus genus, Leuconostoc genus, Acetobacter genus and Streptococcus genus It may be a lactic acid bacteria belonging to the back, preferably Lactobacillus kefiranofaxiens (Lactobacillus kefiranofaciens), Lactobacillus kefir (Lactobacillus kefiri) and at least one selected from the group consisting of Lactobacillus kefirgranum (Lactobacillus kefirgranum) It is preferable that the anti-inflammatory effect and harmful bacteria suppression effect is remarkably superior to other strains, and more preferably, it may include Lactobacillus kefiranofaciens. In addition, in the present invention, yeasts that may be included in the kefir grain include Saccharomyces genus, Lacancea genus, Hanseniasospora genus, Zygotorulaspora genus, It may be yeast belonging to the genus Kluyveromyces, Torula, and Candida, but is not limited thereto.

In the present invention, the microvesicles may be microvesicles derived from cultures of kefir grains.

In the present invention, the culture of the kefir grain is obtained by culturing the kefir grain in a medium, and may collectively refer to all materials obtained through culture, kefir grain, and culture. In addition, the culture may be a liquid culture or a solid culture, the shape is not particularly limited. Here, the type of the medium is also not particularly limited, for example, MRS medium, milk, cheese, and other dairy products may be used.

Specifically, in the present invention, the culture of the kefir grain includes: (a) culturing the kefir grain in a solid medium for 12 to 48 hours first; And (b) primary culturing followed by secondary culturing for 1 to 48 hours in a liquid medium.

In addition, in the present invention, the step (b) of the culture obtained subsequently in step (c) can be carried out in a step of culturing the suspension for a further 1-14 days in a liquid medium. Here, it is preferable to cultivate the suspension for 3 to 7 days during the culture (c), and more preferably, for the culture for 3 to 4 days during the culture (c).

In the present invention, the culture of kefir grains may include extracellular vesicles, and the microvesicles may be naturally or artificially secreted from lactic acid bacteria belonging to the kefir grains, but are not limited thereto. no.

In the present invention, the "microcellular vesicles (extracellular vesicles)" means a small vesicle of a membrane structure secreted from various cells, it is also defined as nanovesicles (nanovesicle). The diameter of the microvesicles is approximately 30 to 1,000 nm, preferably, the average diameter is 100 to 300 nm, and it refers to a vesicle that is released into the extracellular environment by fusion of a polycyst and a plasma membrane.

In the present invention, the amount of the microvesicles contained in the pharmaceutical composition is not particularly limited, but may preferably include 1 X 10 ³ to 1 X 10 ⁹ particles. When the content of the microvesicles is less than 1 X 10 ³ particles, the effect may be negligible, and when it exceeds 1 X 10 ⁹ particles, toxicity may be caused to the human body.

In the present invention, the microvesicles can be obtained by centrifuging a culture of kefir grain. In more detail, in order to obtain the microvesicles, the primary culture of the kefir grain at 100 to 1,000 g at a temperature of 0 to 20 ° C. for 5 minutes to 1 hour; And after the first centrifugation, the supernatant can be used by collecting the supernatant and performing secondary centrifugation for 10 minutes to 1 hour at a temperature of from 0 to 20 ° C below 0 to 1,000 to 5,000 g.

In addition, in the present invention, in order to obtain the microvesicles, the supernatant obtained after the second centrifugation may be used as a supernatant obtained by performing a third centrifugation for 10 minutes to 1 hour at a temperature ranging from 0 to 20 ° C. below 0 to 5,000 to 20,000 g. have.

In addition, the microvesicles in the present invention, the supernatant obtained after the third centrifugation using an ultracentrifuge (ultracentrifuge) to 100,000 ~ 150,000g 4 times centrifugal for 1 hour to 3 hours at a temperature above 0 to 20 ℃ It may be a pellet obtained by separation.

In the present invention, through the four-step centrifugal separation process as described above, microvesicles having excellent anti-inflammatory effects can be obtained with high purity and content.

On the other hand, in the present invention, the microvesicles can be obtained by precipitation from cultures of the kefir grain. More specifically, in order to obtain the microvesicles, the primary centrifugation of the kefir grain culture at 3,000-5,000 g for 5 minutes to 1 hour; After the first centrifugation, 0.5-2N sodium acetate is added to the concentrated culture obtained in a 0.1 to 1.5 volume ratio, preferably 0.1 to 1 volume ratio, more preferably 0.1 to 0.5 volume ratio, and then -10 ° C to 0 ° C Precipitated culture obtained by the step of culturing for 30 to 60 minutes at a temperature can be used.

In addition, in the present invention, the microvesicles may be pellets obtained by secondary centrifugation for 5 minutes to 1 hour at 3,000 to 5,000 g of the precipitated culture obtained after the culture.

In addition, in the present invention, in order to increase the purification efficiency of the microvesicles, the pellet obtained after the second centrifugation is additionally washed with 0.05-0.2N sodium acetate as needed, followed by centrifugation at 3,000-5,000 g for 5 minutes to 1 hour. The pellets thus obtained can be used.

In the present invention, it is preferable that the microvesicles obtained by centrifuging the culture of the kefir grain can enhance the above-mentioned therapeutic effect, but it is possible to use those obtained by a precipitation method in consideration of the efficiency of the manufacturing process.

In the present invention, the microvesicles derived from the culture of kefir grains described above have excellent anti-inflammatory effects and proliferation inhibition and killing effects of harmful bacteria, and can effectively prevent, improve or treat inflammatory diseases.

In the present invention, the inflammatory diseases include sepsis, alopecia areata, acne, allergies, asthma, pemphigus, systemic sclerosis (Scleroderma), conjunctivitis, periodontitis, aphthous stomatitis, rhinitis, otitis media, pharyngitis, tonsillitis, chronic thyroiditis (Hashimoto 氏) Hashimoto's thyroiditis, pneumonia, silicosis (silicosis), asbestos (asbestosis), gastric ulcer, gastritis, hemorrhoids, gout, ankylosing spondylitis, rheumatoid arthritis, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid Arthritis, peri-arthritis, tendonitis, hayitis, tendonitis, myositis, hepatitis, cystitis, nephritis, post-streptococcal glomerulonephritis, atherosclerosis, degenerative arthritis, Sjogren's syndrome, multiple sclerosis (multiple sclerosis), polymyositis, dermatomyositis, nodular polyarteritis (Polya rteritis nodosa, Ankylosing spondylitis, Fibromyalgia syndrome, Temporal arteritis, Guilian-Barre syndrome, Autoimmune hemolytic anemia, Autoimmune encephalomyelitis, It can be selected from the group consisting of Myasthenia gravis, Grave's disease and inflammatory back pain.

According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for the prevention or treatment of intestinal diseases comprising microvesicles derived from kefir grains as an active ingredient.

In the present invention, the kefir grain essentially includes lactic acid bacteria, and may further include yeast, and the lactic acid bacteria include Lactobacillus genus, Lactococcus genus, and Leuconostoc. It may be a lactic acid bacterium belonging to the genus, Acetobacter genus, Streptococcus genus, etc., preferably Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefiri Containing at least one selected from the group consisting of num (Lactobacillus kefirgranum) is preferred because it has a remarkably excellent anti-inflammatory effect, improved bowel activity, improved intestinal immunity, and inhibitory effect of harmful bacteria in the intestine, more preferably lactobacillus kepi Lanofaxiens (Lactobacillus kefiranofaciens) may include. In addition, in the present invention, the yeast is Saccharomyces (Saccharomyces) genus, Lacancea (Lachancea) genus, Hanseniasospora (Hanseniasospora) genus, Zygotorulaspora (Zygotorulaspora) genus, Kluyveromyces (Kluyveromyces) It may be yeast belonging to the genus, the genus Torula and the genus Candida, but is not limited thereto.

In the present invention, the microvesicles may be microvesicles derived from cultures of kefir grains.

In the present invention, the culture of the kefir grain is obtained by culturing the kefir grain in a medium, and may collectively refer to all materials obtained through culture, kefir grain, and culture. In addition, the culture may be a liquid culture or a solid culture, the shape is not particularly limited. Here, the type of the medium is also not particularly limited, for example, MRS medium, milk, cheese, and other dairy products may be used.

Specifically, in the present invention, the culture of the kefir grain includes: (a) culturing the kefir grain in a solid medium for 12 to 48 hours first; And (b) primary culturing followed by secondary culturing for 1 to 48 hours in a liquid medium.

In addition, in the present invention, the step (b) of the culture obtained subsequently in step (c) can be carried out in a step of culturing the suspension for a further 1-14 days in a liquid medium. Here, it is preferable to cultivate the suspension for 3 to 7 days during the culture (c), and more preferably, for the culture for 3 to 4 days during the culture (c).

In the present invention, the culture of kefir grains may include extracellular vesicles, and the microvesicles may be naturally or artificially secreted from lactic acid bacteria belonging to the kefir grains, but are not limited thereto. no.

In the present invention, the amount of the microvesicles contained in the pharmaceutical composition is not particularly limited, but may preferably include 1 X 10 ³ to 1 X 10 ⁹ particles. When the content of the microvesicles is less than 1 X 10 ³ particles, the effect may be negligible, and when it exceeds 1 X 10 ⁹ particles, toxicity may be caused to the human body.

In the present invention, the microvesicles can be obtained by centrifuging a culture of kefir grain. In more detail, in order to obtain the microvesicles, the primary culture of the kefir grain at 100 to 1,000 g at a temperature of 0 to 20 ° C. for 5 minutes to 1 hour; And after the first centrifugation, the supernatant can be used by collecting the supernatant and performing secondary centrifugation for 10 minutes to 1 hour at a temperature of from 0 to 20 ° C below 0 to 1,000 to 5,000 g.

In addition, in the present invention, in order to obtain the microvesicles, the supernatant obtained after the second centrifugation may be used as a supernatant obtained by performing a third centrifugation for 10 minutes to 1 hour at a temperature ranging from 0 to 20 ° C. below 0 to 5,000 to 20,000 g. have.

In addition, the microvesicles in the present invention, the supernatant obtained after the third centrifugation using an ultracentrifuge (ultracentrifuge) to 100,000 ~ 150,000g 4 times centrifugal for 1 hour to 3 hours at a temperature above 0 to 20 ℃ It may be a pellet obtained by separation.

In the present invention, through the four-step centrifugal separation process as described above, it is possible to obtain anti-inflammatory, defecation activity improvement, intestinal immunity enhancement, proliferation inhibition and killing effects of harmful bacteria in the gut with high purity and content.

On the other hand, in the present invention, the microvesicles can be obtained by precipitation from cultures of the kefir grain. More specifically, in order to obtain the microvesicles, the primary centrifugation of the kefir grain culture at 3,000-5,000 g for 5 minutes to 1 hour; After the first centrifugation, 0.5-2N sodium acetate is added to the concentrated culture obtained in a 0.1 to 1.5 volume ratio, preferably 0.1 to 1 volume ratio, more preferably 0.1 to 0.5 volume ratio, and then -10 ° C to 0 ° C Precipitated culture obtained by the step of culturing for 30 to 60 minutes at a temperature can be used.

In addition, in the present invention, the microvesicles may be pellets obtained by secondary centrifugation for 5 minutes to 1 hour at 3,000 to 5,000 g of the precipitated culture obtained after the culture.

In addition, in the present invention, in order to increase the purification efficiency of the microvesicles, the pellet obtained after the second centrifugation is additionally washed with 0.05-0.2N sodium acetate as needed, followed by centrifugation at 3,000-5,000 g for 5 minutes to 1 hour. The pellets thus obtained can be used.

In the present invention, it is preferable that the microvesicles obtained by centrifuging the culture of the kefir grain can enhance the above-mentioned therapeutic effect, but it is possible to use those obtained by a precipitation method in consideration of the efficiency of the manufacturing process.

In the present invention, the microvesicles derived from the culture of kefir grains described above have excellent anti-inflammatory effects, improved bowel activity, improved intestinal immunity, proliferation inhibition and killing effects of harmful bacteria in the intestine, and can effectively prevent, improve or treat intestinal diseases. .

In the present invention, the bowel disease may be irritable bowel syndrome or inflammatory bowel disease.

In addition, in the present invention, the irritable bowel syndrome may be diarrhea predominant irritable bowel syndrome (d-IBS), constipation predominant irritable bowel syndrome, or diarrhea-constipation mixed irritable bowel syndrome.

In addition, in the present invention, the inflammatory bowel disease may be selected from the group consisting of Crohn's disease, intestinal lesion accompanying Behcet's disease, ulcerative colitis, hemorrhagic rectal ulcer, and ileal cystitis.

According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating mental disorders including kefir grain-derived microvesicles as an active ingredient.

In the present invention, the kefir grain essentially includes lactic acid bacteria, and may further include yeast, and the lactic acid bacteria include Lactobacillus genus, Lactococcus genus, and Leuconostoc. It may be a lactic acid bacterium belonging to the genus, Acetobacter genus, Streptococcus genus, etc., preferably Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefiri Containing one or more selected from the group consisting of num (Lactobacillus kefirgranum) has a remarkably excellent effect of suppressing the expression of cytokines due to stress compared to other strains, and more preferably, Lactobacillus kefiranofaciens It may include. In addition, in the present invention, the yeast is Saccharomyces (Saccharomyces) genus, Lacancea (Lachancea) genus, Hanseniasospora (Hanseniasospora) genus, Zygotorulaspora (Zygotorulaspora) genus, Kluyveromyces (Kluyveromyces) It may be yeast belonging to the genus, the genus Torula and the genus Candida, but is not limited thereto.

In the present invention, the microvesicles may be microvesicles derived from cultures of kefir grains.

In the present invention, the culture of the kefir grain is obtained by culturing the kefir grain in a medium, and may collectively refer to all materials obtained through culture, kefir grain, and culture. In addition, the culture may be a liquid culture or a solid culture, the shape is not particularly limited. Here, the type of the medium is also not particularly limited, for example, MRS medium, milk, cheese, and other dairy products may be used.

Specifically, in the present invention, the culture of the kefir grain includes: (a) culturing the kefir grain in a solid medium for 12 to 48 hours first; And (b) primary culturing followed by secondary culturing for 1 to 48 hours in a liquid medium.

In addition, in the present invention, the step (b) of the culture obtained subsequently in step (c) can be carried out in a step of culturing the suspension for a further 1-14 days in a liquid medium. Here, it is preferable to cultivate the suspension for 3 to 7 days during the culture (c), and more preferably, for the culture for 3 to 4 days during the culture (c).

In the present invention, the culture of kefir grains may include extracellular vesicles, and the microvesicles may be naturally or artificially secreted from lactic acid bacteria belonging to the kefir grains, but are not limited thereto. no.

In the present invention, the amount of the microvesicles contained in the pharmaceutical composition is not particularly limited, but may preferably include 1 X 10 ³ to 1 X 10 ⁹ particles. When the content of the microvesicles is less than 1 X 10 ³ particles, the effect may be negligible, and when it exceeds 1 X 10 ⁹ particles, toxicity may be caused to the human body.

In the present invention, the microvesicles can be obtained by centrifuging a culture of kefir grain. In more detail, in order to obtain the microvesicles, the primary culture of the kefir grain at 100 to 1,000 g at a temperature of 0 to 20 ° C. for 5 minutes to 1 hour; And after the first centrifugation, the supernatant can be used by collecting the supernatant and performing secondary centrifugation for 10 minutes to 1 hour at a temperature of from 0 to 20 ° C below 0 to 1,000 to 5,000 g.

In addition, in the present invention, in order to obtain the microvesicles, the supernatant obtained after the second centrifugation may be used as a supernatant obtained by performing a third centrifugation for 10 minutes to 1 hour at a temperature ranging from 0 to 20 ° C. below 0 to 5,000 to 20,000 g. have.

In addition, the microvesicles in the present invention, the supernatant obtained after the third centrifugation using an ultracentrifuge (ultracentrifuge) to 100,000 ~ 150,000g 4 times centrifugal for 1 hour to 3 hours at a temperature above 0 to 20 ℃ It may be a pellet obtained by separation.

In the present invention, microvesicles having an excellent effect of suppressing the expression of cytokines due to stress can be obtained with high purity and content through the four-step centrifugation process described above.

On the other hand, in the present invention, the microvesicles can be obtained by precipitation from cultures of the kefir grain. More specifically, in order to obtain the microvesicles, the primary centrifugation of the kefir grain culture at 3,000-5,000 g for 5 minutes to 1 hour; After the first centrifugation, 0.5-2N sodium acetate is added to the concentrated culture obtained in a 0.1 to 1.5 volume ratio, preferably 0.1 to 1 volume ratio, more preferably 0.1 to 0.5 volume ratio, and then -10 ° C to 0 ° C Precipitated culture obtained by the step of culturing for 30 to 60 minutes at a temperature can be used.

In addition, in the present invention, the microvesicles may be pellets obtained by secondary centrifugation for 5 minutes to 1 hour at 3,000 to 5,000 g of the precipitated culture obtained after the culture.

In addition, in the present invention, in order to increase the purification efficiency of the microvesicles, the pellet obtained after the second centrifugation is additionally washed with 0.05-0.2N sodium acetate as needed, followed by centrifugation at 3,000-5,000 g for 5 minutes to 1 hour. The pellets thus obtained can be used.

In the present invention, it is preferable that the microvesicles obtained by centrifuging the culture of the kefir grain can enhance the above-mentioned therapeutic effect, but it is possible to use those obtained by a precipitation method in consideration of the efficiency of the manufacturing process.

Recent studies have shown that physical and psychological stress increases IL-6 levels, resulting in depressive symptoms (Korean J Psychopharmacol 2000; 11 (4): 304-312). However, in the present invention, the microvesicles derived from the culture of kefir grains may decrease the secretion of IL-6, a stressful cytokine caused by oxidative stress in adrenal medulla cells, and thus, anxiety, depression, mood disorders, It can effectively prevent, ameliorate or treat mental disorders such as insomnia, obsessive-compulsive disorder or stress.

On the other hand, in the present invention, "prevention" may include, without limitation, any action that blocks, suppresses or delays the symptoms of the diseases listed above using the pharmaceutical composition of the present invention.

In addition, in the present invention, "treatment" may include, without limitation, any action in which the symptoms of the diseases listed above are improved or beneficially altered using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized for humans.

The pharmaceutical composition of the present invention is not limited to these, but can be used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. have. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. when administered orally, and buffers, preservatives, and analgesics for injections. Agents, solubilizers, isotonic agents, stabilizers, etc. can be used in combination, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. may be prepared, and in the case of injection, unit dosage ampoules or multiple doses may be prepared. have. Others can be formulated as solutions, suspensions, tablets, capsules, and sustained release preparations.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be further included.

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, This includes sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, epidural, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention may vary depending on various factors including the activity, age, weight, general health, sex, diet, administration time, administration route, discharge rate, drug combination and severity of a specific disease to be prevented or treated. It may vary, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, dosage form, administration route and duration, but can be appropriately selected by those skilled in the art, and 0.0001 to 50 mg / kg per day, or It can be administered at 0.001 to 50mg / kg. The administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way. Pharmaceutical compositions according to the invention may be formulated as pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions.

According to another embodiment of the present invention, the present invention relates to a food composition for preventing or improving an inflammatory disease comprising kefir grain-derived microvesicles as an active ingredient.

According to another embodiment of the present invention, the present invention relates to a food composition for preventing or improving intestinal diseases comprising microvesicles derived from kefir grains as an active ingredient.

 According to another embodiment of the present invention, the present invention relates to a food composition for preventing or improving mental disorders including kefir grain-derived microvesicles as an active ingredient.

In the food composition for each use of the present invention, specific details regarding the kefir grain, microvesicle, inflammatory disease, intestinal disease, and mental disorder are duplicated as described in the pharmaceutical composition, and detailed description thereof will be omitted below.

In the present invention, "improvement" means any act of using the food composition of the present invention to improve or benefit the symptoms of the diseases listed above.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, cookies, rice cakes, breads, and the like. Since the food composition of the present invention is composed of plant extracts having little toxicity and side effects, it can be safely used even for a long period of time for prevention purposes.

When the microvesicles of the present invention are included in the food composition, the amount may be added at a rate of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in the form of a beverage, there are no particular limitations other than containing the food composition in an indicated ratio, and it may contain various flavoring agents or natural carbohydrates, etc., as additional components, like a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, sucrose, etc., and common sugars such as polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

Other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like.

These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

According to another embodiment of the present invention, the present invention relates to a cosmetic composition for preventing or improving an inflammatory disease comprising kefir grain-derived microvesicles as an active ingredient.

In addition, in the present invention, the kefir grains, preferably the microvesicles derived from the culture of the kefir grains, have excellent anti-inflammatory effects and can effectively prevent or improve inflammatory diseases such as edema, dermatitis, allergies or atopy.

In the cosmetic composition of the present invention, specific details regarding the kefir grains and microvesicles are overlapped with those described in the pharmaceutical composition, and detailed description thereof will be omitted below.

In addition, the cosmetic composition in the present invention is a lotion, nutrient lotion, nutrient essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen Cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, hair loss agent {for cosmetics}, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine Oil, bath soap, water soap, beauty soap, shampoo, hand cleaner (hand cleaner), medicinal soap (non-medical use), cream soap, facial wash, whole body cleaner, scalp cleaner, hair rinse, cosmetic soap, tooth whitening gel, toothpaste And the like. To this end, the composition of the present invention may further include a solvent or a suitable carrier, excipient or diluent, which is commonly used in the preparation of cosmetic compositions.

The type of the solvent that can be further added in the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof can be used, and purified water, oil, wax as a carrier, excipient, or diluent , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like. In addition, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as necessary.

Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil can be used as the oil, and waxes include beeswax, mellow, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin. Can be used.

As the fatty acid, stearic acid, linoleic acid, linolenic acid, and oleic acid may be used, and as the fatty acid alcohol, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used. As the fatty acid ester, isopropyl myristate, isopropyl palmitate, and butyl stearate can be used. As the surfactant, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred if possible.

In addition, it may include a hygroscopic agent, a thickener, an antioxidant, etc., which are widely known in the cosmetic field, and their types and amounts are as known in the art.

The composition provided by the present invention has excellent anti-inflammatory effect, and thus can effectively prevent, improve or treat various inflammatory diseases.

In addition, the composition provided by the present invention has an excellent effect of suppressing the expression of inflammatory factors in colon cells, improves bowel movement, improves immunity in the intestine, suppresses proliferation of harmful bacteria in the intestine and induces death, effectively preventing and improving intestinal diseases Or it can be treated.

In addition, the composition provided by the present invention can significantly reduce the expression level of IL-6 due to physical or mental stress, thereby effectively preventing, improving or treating stress-based mental disorder.

Figure 1 (a) shows the results of measuring the number of particles and the average size of the microvesicles contained in the concentrated culture obtained by centrifuging the Lactobacillus kefir culture in Experimental Example 1 with NTA.
Figure 1 (b) shows the results of measuring the number of particles and the average size of the microvesicles contained in the concentrated culture obtained by centrifuging the Lactobacillus kefiranofaxiens culture in Experimental Example 1 with NTA.
Figure 1 (c) shows the result of measuring the number of particles and the average size of the microvesicles contained in the concentrated culture obtained by centrifuging the Lactobacillus kefir granum culture in Experimental Example 1 with NTA.
Figure 2 (a) shows the content of the protein contained in the culture of Lactobacillus kefir, Lactobacillus kefiranofaxiens and Lactobacillus kefirgranum in each culture time in Experimental Example 2.
Figure 2 (b) is the result of measuring the number of particles of the microvesicles contained in the culture of Lactobacillus kefir, Lactobacillus kefiranofaxiens, and Lactobacillus kefirgranum in Experimental Example 2 with NTA. It is shown.
Figure 3 shows the results of measuring the number of particles of microvesicles according to the separation method from the culture of Lactobacillus kefir, Lactobacillus kefiranofaxiens and Lactobacillus kefirgranum in Experimental Example 3 with NTA.
Figure 4 shows the results by measuring the mRNA expression levels of IL-8 and TNF-α after treatment with the microvesicles according to the present invention to the inflammation-induced colon cell line CaCo-2 in Experimental Example 5.
Figure 5 shows the results of measuring the expression level of IL-6 after stimulation with LPS after treating the microvesicles according to the present invention to macrophages (Raw246.7 cells) in Experimental Example 6.
Figure 6 shows the results of measuring the expression level of TNF-α after stimulation with LPS after treating the microvesicles according to the present invention to macrophages (Raw246.7 cells) in Experimental Example 6.
7 shows the result of measuring the expression level of IL-8 after treating the microvesicles according to the present invention after treating TNF-α to Caco-2 cells in Experimental Example 7.
8 shows the result of measuring the expression level of TNF-α after treating the microvesicles according to the present invention after treating TNF-α to Caco-2 cells in Experimental Example 7.
9 shows the result of measuring the secretion rate of IL-8 compared to the negative control after treating the microvesicles according to the present invention after treating TNF-α to Caco-2 cells in Experimental Example 7.
Figure 10 shows a photograph of colon tissue after microvesicle treatment according to the present invention in a mouse model of TNBS-induced colitis in Experimental Example 8.
Figure 11 shows the results of histological evaluation of colon tissue after microvesicle treatment according to the present invention in a mouse model of TNBS-induced colitis in Experimental Example 8.
Figure 12 shows the results of evaluating fecal homeostasis after treatment of the microvesicles (123_v) derived from the mixed strain culture according to the present invention in the TNBS-induced colitis mouse model in Experimental Example 8.
13 shows the results of evaluating the degree of colon bleeding after treatment of the microvesicles (123_v) derived from the mixed strain culture according to the present invention in the TNBS-induced colitis mouse model in Experimental Example 8.
Figure 14 shows the results of evaluating fecal homeostasis after treatment of the culture-derived microvesicles (1_v) of Lactobacillus kefirgranum according to the present invention in a mouse model of TNBS-induced colitis in Experimental Example 8.
Figure 15 shows the results of evaluating the degree of colon bleeding after treatment of the culture-derived microvesicles (1_v) of Lactobacillus kefirgranum according to the present invention to the TNBS-induced colitis mouse model in Experimental Example 8.
Figure 16 shows the results of measuring the change in MPO activity after microvesicle treatment according to the present invention in TNBS-induced colitis mouse model in Experimental Example 8.
Figure 17 shows the results of evaluating the degree of colon bleeding after treating the microvesicles according to the present invention in the mouse model of acetic acid-induced colitis in Experimental Example 9.
18 shows the results of measuring the number of viable bacteria after treatment with the microvesicles according to the present invention in Staphylococcus epidermidis in Experimental Example 10.
19 shows the results of measuring the expression level of IL-6 after treatment with microvesicles according to the present invention after treating hydrogen peroxide (H ₂ O ₂ ) on adrenal medulla derived PC-12 cells in Experimental Example 11.
Figure 20 (a) shows the results of measuring the cell viability after treatment with the microvesicles according to the invention in 3T3-L1 cells in Experimental Example 12.
20 (b) shows the results of measuring cell viability after treatment with microvesicles according to the present invention in CaCo-2 cells in Experimental Example 12.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

[Experimental Example 1] Kefir grain culture-derived microvesicle size and particle number confirmation

Each of Lactobacillus kefiranofaciens, Lactobacillus kefiri, and Lactobacillus kefirgranum contained in kefir grains is streaked at 37 ° C. by streaking in solid MRS medium. After incubation for 24 hours, the cells were suspended in a liquid MRS medium for 24 hours on a small scale. After that, the OD value was adjusted and the suspension was cultured on a large scale in a liquid MRS medium. On the 4th day of cultivation, the culture was collected, and the culture was centrifuged at 300 g for 10 minutes at 4 ° C, the supernatant was centrifuged at 1,200g for 20 minutes at 4 ° C, and the supernatant was taken for 10,000 g 4 After centrifugation at 30 ° C for 30 minutes, the supernatant was taken and centrifuged at 110,000 g for 1 hour and 10 minutes at 110,000 g using an ultracentrifuge, and then the supernatant was removed to suspend the precipitate with PBS. The number of particles and the average size of the microvesicles contained in 1 ml of the concentrated culture of lactic acid bacteria thus obtained was measured using NTA (Nanosight NS300), and the results are shown in FIGS. 1 (a) to (c) and Table 1 below. .

Strain Total count
(particles / ml) Lactobacillus kefiranofaxiens 6.0 X 10 ¹¹ Lactobacillus kefir 3.0 X 10 ¹¹ Lactobacillus kefir granum 3.0 X 10 ¹¹

As shown in (a) to (c) of FIG. 1 and Table 1, it was confirmed that microvesicles were included in an average of 4.5 X 10 ¹¹ particles per 1 ml of lactic acid bacteria culture, and the average size was 120 to 190 nm. It was confirmed by.

[Experimental Example 2] Change in yield of microvesicles by culture period of lactic acid bacteria

Each of the Lactobacillus kefiranofaxiens, Lactobacillus kefir, and Lactobacillus kefirgranum strains contained in Kefir grains is streaked in solid MRS medium and incubated at 37 ° C. for 24 hours, followed by liquid MRS medium. Incubated in suspension on a small scale for 24 hours. After that, the OD value was adjusted and the suspension was cultured on a large scale in a liquid MRS medium. Cultures were collected on day 1, day 2, day 3, day 4 and day 7 of culture, centrifuged with an ultracentrifuge to discard the supernatant, and the concentrated culture of lactic acid bacteria was recovered. NTA (Nanosight NS300) was used to measure the particle number and protein content of microvesicles contained in 1 ml of the culture, and the results are shown in FIGS. 2 (a) and 2 (b).

As shown in (a) and (b) of FIG. 2, as a result of confirming microvesicles by protein and particle number for each culture day, it was confirmed that the particle number method compared to the protein has a certain pattern for each culture day. On the basis of the particle counting method, it was confirmed that all three types of lactic acid bacteria contained the most microvesicles in the culture on the fourth day of culture.

[Experimental Example 3] Yield change by separation method of microvesicles

Microvesicles were separated from the day 4 culture obtained in Experimental Example 1 using the following three methods.

1. Separation of microvesicles using ultracentrifuge (ultracentrifuge method)

The culture was centrifuged at 300 g at 4 ° C. for 10 minutes, the supernatant was centrifuged at 1,200 g at 4 ° C. for 20 minutes, and the supernatant was taken at 10,000 g and centrifuged at 4 ° C. for 30 minutes, The supernatant was taken and centrifuged for 1 hour and 10 minutes at 4 ° C at 110,000 g using an ultracentrifuge, and then the supernatant was removed to suspend the precipitate with PBS.

2. Separation of microvesicles using ExoQuick-TC ^TM exosome precipitation solution (Exoquick method)

The culture was centrifuged at 3,000 g for 15 minutes, and the exosome precipitation solution of ExoQuick-TC ^TM was added at a rate of 2 ml per 10 ml of the culture, and then cultured overnight at 4 ° C. After incubation, the supernatant was removed after centrifugation at 1,500 g for 30 minutes, and the supernatant was removed after centrifugation at 1,500 g for 30 minutes, and the precipitate was suspended with PBS.

3. Separation of microvesicles using precipitation method (precipitation method)

The culture was centrifuged at 5,000 g for 10 minutes, 1N sodium acetate was added at a rate of 1 ml per 10 ml of culture, and then cultured on ice for 45 minutes. After incubation, after centrifugation at 5,000 g for 10 minutes, the supernatant was removed and the precipitate was washed with 0.1N sodium acetate. Thereafter, after centrifugation at 5,000 g for 10 minutes, the supernatant was removed and the pellet was suspended with PBS.

The number of particles of microvesicles contained in the precipitates obtained by the above three methods was measured using NTA (Nanosight NS300), and the results are shown in FIG. 3.

As shown in Figure 3, it can be seen that by using a method using an ultracentrifuge, microvesicles can be obtained with the highest yield from lactic acid bacteria culture.

[Experimental Example 4] Acquisition of microvesicles derived from kefir grains

After obtaining the culture by culturing a mixed strain of Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefirgranum in the same manner as in Experimental Example 1 above , The microvesicles were separated by the method using the ultracentrifuge of Experimental Example 3.

[Experimental Example 5] Confirmation of anti-inflammatory effect of microvesicles derived from kefir grains (1)

After plating the inflammation-induced colon cell line CaCo-2 with 20 ng of TNF-α at 1 X 10 ⁶ cells per well in a 12-well plate, each microvesicle separated using an ultracentrifuge in Experimental Example 3 was 1 X. Treatment was carried out for 24 hours in the amount of 10 ³ , 1 X 10 ⁶ , 1 X 10 ⁹ particles. Thereafter, after 48 hours of culture, RNA from each group of cells is extracted, and then complementary DNA (cDNA) is synthesized using an intron premix and real-time PCR (RT-PCR) is performed using the same. Was performed. The expression levels of IL-8 and TNF-α were measured as inflammatory factors in cells of each treatment group, and the results are shown in FIG. 4.

As shown in FIG. 4, when the microvesicles derived from the lactic acid bacteria culture according to the present invention are treated, it can be seen that the expression levels of IL-8 and TNF-α are significantly reduced. In particular, the microvesicles derived from Lactobacillus kefiranofaxiens have anti-inflammatory effect. You can see something outstanding.

[Experimental Example 6] Confirmation of anti-inflammatory effect of microvesicles derived from kefir grains (2)

After culturing the macrophages of the mouse Raw246.7 cells in DMEM medium containing 10% FBS and 1% penicillin-streptomycin for 48 hours, the microvesicles obtained in Experimental Example 4 were 200 µg / ml (1 X 10 ⁹ nano Particles / ml) was incubated for 24 hours, LPS 100ng / ml was treated for 6 hours, and then the expression levels of IL-6 and TNF-α were measured using RT-PCR and ELISA to determine the results. 5 and 6. However, as a positive control, curcumin was treated in an amount of 10 μM.

As shown in Figures 5 and 6, when treated with microvesicles according to the present invention to macrophages and inducing inflammation using LPS, IL-6 and TNF-α compared to those not treated or curcumin treated It was confirmed that the expression level was significantly reduced to more than half.

[Experimental Example 7] Confirmation of anti-inflammatory effect of kefir grain-derived microvesicles (3)

5% CO ₂ -95% air in an incubator at 37 ° C. by adding 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, 0.1 mmol / l non-essential amino acid, 1 mmol / l sodium pyruvate to the MEM medium Caco-2 cells were cultured with the composition. When the cultured cells grew to 80% confluency, cells were removed using 0.25% trypsin, and 24 wells were inoculated with 2 X 10 ⁴ cells per well. Subsequently, after treating TNF-α in each well with an amount of 20 ng / ml for 6 hours, the microvesicles obtained in Experimental Example 4 were treated with an amount of 200 μg / ml (1 X 10 ⁹ nanoparticles / ml) 24 Incubated for hours. The expression level of IL-8 and TNF-α and the degree of secretion of IL-8 in the microvesicle treated group compared to the negative control group were measured using RT-PCR and ELISA (100 μg / ml), and the results are shown in FIGS. 7 to It is shown in 9.

As shown in Figures 7 to 9, when the intestinal epithelial cells are treated with TNF-α to induce inflammation, the microvesicles according to the present invention are treated to significantly reduce the mRNA levels of the inflammatory factors IL-8 and TNF-α. In addition, it was confirmed that the amount of IL-8 secretion was also significantly reduced.

[Experimental Example 8] Confirm the anti-inflammatory effect of kefir grain-derived microvesicles (4)

In BALB / c mice, colitis was induced with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Specifically, after anesthetizing the mouse with ether, a solution obtained by mixing 2.5 g of a TNBS (2,4,6-Trinitrobenzene sulfonic acid) solution in 50% ethanol was injected into the large intestine through the anus using a 1 ml syringe with a round end. It was administered by ㎖ each and held vertically for 30 seconds to cause inflammation. On the other hand, 0.1 ml of physiological saline was orally administered to the normal group. Thereafter, the microvesicles obtained in Experimental Example 4 were administered orally at a dose of 10 µg / mari or 1 mg / mari once daily for 3 days from the next day. The large intestine from the middle appendix to the site just before the anus was removed. However, as a positive control, prednisolone, a treatment for colitis, was administered orally in an amount of 2 mg / kg.

(1) Organizational appearance evaluation

After H & E staining of the extracted colon tissues, cross-sectional photographs are shown in FIG. 10, and the length and appearance of the colons were observed, and scored according to the criteria in Table 2 below (Hollenbach et al., 2005 criteria for colitis). The results are shown in FIG. 11.

Macroscopic Score standard 0 No ulcers and inflammation were found One Redness without bleeding was found 2 Hyperulcerative ulcers found 3 Sheep and inflammation found only in one place 4 Ulcers and inflammation found in 2 or more locations 5 Ulcer is enlarged to 2 cm or more

As shown in FIG. 10, in the large intestine of the colitis mouse model, severe epithelial necrosis, bleeding, infiltration of inflammatory cells, edema, and ulcers were observed, but when the microvesicles according to the present invention were administered, the symptoms of colitis described above were concentration-dependent. It was confirmed that it was significantly relaxed.

In addition, as shown in FIG. 11, when the microvesicles according to the present invention were administered to the colitis mouse model, bleeding in the colon was decreased depending on the concentration, and such a therapeutic effect was to administer prednisolone in a large amount of 2 mg / kg as a positive control. It was confirmed that it was superior to one case.

(2) DAI evaluation

DAI (disease activity index) is a method for scoring symptoms caused by TNBS causing acute colitis, starting from the time of providing the microvesicles according to the present invention, and providing the concentration and color of the stool at the same time during the day. It was measured by checking. Fecal homeostasis (consistency) and bleeding were measured, and the results are shown in FIGS. 12 to 15. However, the evaluation criteria of DAI are shown in Table 3 below. When evaluating DAI, in addition to the microvesicles (123_v) derived from the culture of the mixed strain obtained in Experimental Example 4, the microvesicles (1_v) derived from the Lactobacillus kefir granum culture obtained using the ultracentrifuge in Experimental Example 3 were administered. To further experiment.

Stool consistency Stool bleeding 0 Formed 0 Normal color 2 Loose stool 2 fecal occult blood test positive 4 Diarrhea 4 Gross bleeding

As shown in Figs. 12 to 15, when the culture microvesicles (123_v) of the mixed strain according to the present invention or the microvesicles (1_v) derived from kefir granum cultures as a lactobaciller is administered as a positive control, as a positive control It was confirmed that the bowel activity was improved by lowering the DAI value to a level equal to or greater than that of prednisolone.

(3) MPO activity evaluation

In addition, the MPO ELISA kit (Hycult Biotech, HK105) was used to measure the activity of Myeloperoxidase (MPO), an index of inflammation in the extracted colon tissue, and the results are shown in FIG. 16. However, the MPO activity is 100 μl of the supernatant obtained in advance by centrifuging the intestinal tissue homogeneously crushed with a homogenizer for 15 minutes and a standard solution prepared in advance in the wells to which the antibody is attached. , Sealed with a cover to prevent air from entering the plate, and then reacted for 1 hour at room temperature. After the reaction was completed for 1 hour, washing was performed 4 times using a washing buffer so that the washing buffer was completely removed. Thereafter, 100 μl of the tracer solution was added to each well, and the cover was attached to react for 1 hour at room temperature and washed 4 times. Then, the diluted streptavidin-peroxidase solution was added to every well in 100 μl, and a cover was attached to react at room temperature for 1 hour. After the reaction was completed, each well was washed 4 times, 100 μl of TMB substrate was added to all wells, and then reacted for 30 minutes under shading, and then 100 μl of the same amount of stop solution was added. After that, the absorbance was measured at 450 nm to obtain the MPO activity value of the sample using a standard curve made of a standard solution.

As shown in FIG. 16, when the microvesicles according to the present invention were administered to the colitis mouse model, it was confirmed that MPO activity was significantly decreased to a level equal to or higher than that of prednisolone as a positive control.

[Experimental Example 9] Confirmation of anti-inflammatory effect of kefir grain-derived microvesicles (5)

Irritable bowel syndrome was induced by acetic acid in BALB / c mice. Specifically, after anesthetizing the mouse with ether, 0.1 ml of 5% acetic acid was administered into the large intestine through the anus using a syringe with a rounded end of 1 ml, and maintained vertically for 30 seconds to cause irritable bowel syndrome. On the other hand, 0.1 ml of physiological saline was orally administered to the normal group. Subsequently, the microvesicles obtained in Experimental Example 4 were administered orally at a dose of 10 µg / maritime once daily for 3 days from the next day. Starting from the time of providing the microvesicles according to the present invention, the color of the stool is checked at the same time every day during the provision, and the bleeding degree of the colon is measured based on the criteria shown in Table 3, and the results are shown in FIG. 17. Did. However, as a positive control group, prednisolone (2 mg / kg), Lactobacillus kefiranofaciens, Lactobacillus kefiri, and mixed strains of Lactobacillus kefirrinum (Lactobacillus kefirgranum) (5 X 10 ⁷ CFU), a commercially available lactic acid bacteria product (5 X 10 ⁷ CFU) and a commercially available lactic acid bacteria product (5 X 10 ⁷ CFU) and the microvesicles (10 µg / mari) of the present invention were administered orally.

As shown in FIG. 17, when the microvesicles according to the present invention were treated with the mouse model of acetic acid-induced irritable bowel syndrome, the mixed strains derived from the microvesicles themselves were administered, or the lactic acid bacteria previously sold on the market were significantly remarkable. It was confirmed that the bleeding in the colon was reduced. In addition, it was confirmed that when the microvesicles according to the present invention were treated with lactic acid bacteria that were commercially available on the market, the degree of colon bleeding reduction was further increased.

[Experimental Example 10] Confirmation of antibacterial effect of microvesicles derived from kefir grains

In order to confirm the antimicrobial effect of kefir grain-derived microvesicles against harmful bacteria in the intestine, Staphylococcus epidermidis belonging to the harmful bacteria in the intestine was cultured in a liquid nutrient medium for 24 hours, and then the experimental example The microvesicles obtained in 4 were treated with an amount of 500 µg / ml, further cultured for 3 hours at 37 ° C., and the number of viable bacteria was measured and the results are shown in FIG. 18. However, distilled water was treated as a negative control, and penicillin was treated as a positive control in an amount of 10 μg / ml.

As shown in Figure 18, when treating the microvesicles according to the present invention it can be seen that inhibiting the proliferation of harmful bacteria in the intestine, Staphylococcus epidermidis, and inducing death, at the same level as when treating the microvesicles according to the present invention. there was.

[Experimental Example 11] Confirmation of the stress relieving effect of microvesicles derived from kefir grains

In order to evaluate the stress relieving effect of kefir grain-derived microvesicles according to the present invention, each well of a 24-well plate contains 10% serum (FBS: Fetal Bovine Serum) and 1% penicillin / streptomycin antibiotic MEM (Minimal Essential Medium) was dispensed, and each well was inoculated with adrenal medulla-derived PC-12 cells 5 X 10 ⁴ cells, followed by treatment with hydrogen peroxide (H ₂ O ₂ ) for 1 hour to induce IL-6 expression. . Subsequently, the microvesicles obtained in Experimental Example 4 were treated in amounts of 0.1 μg / ml, 1 μg / ml, 10 μg / ml, and 100 μg / ml, respectively, and the expression level of IL-6 was measured using ELISA. The results are shown in Figure 19.

As shown in FIG. 19, when the microvesicles according to the present invention were treated, the expression level of IL-6 was significantly reduced compared to the untreated group, and it was confirmed that the degree of reduction increased in proportion to the concentration.

Through this, when using kefir grain-derived microvesicles according to the present invention, it was obviously predicted that mental disorders such as depression can also be prevented or treated by significantly reducing the expression level of IL-6 due to stress.

[Experimental Example 12] Confirmation of the safety of microvesicles derived from kefir grains

In order to confirm the safety of the microvesicles derived from kefir grains according to the present invention, the microvesicles obtained by using the ultracentrifuge in Experimental Example 3 in 3T3-L1 and CaCo-2 normal cells were 1 X 10 ³ to 1 X 10 ⁹ After treating with the amount of particles, dilute the MTS reagent (Promega, USA) and the cell's basic medium (FBS free, 1% p / s) at a ratio of 1: 5 and dispense 500 µl per well for 1 hour and 30 minutes. After the reaction, the absorbance was measured at a wavelength of 490 nm to evaluate the change in cell viability, and the results are shown graphically in FIGS. 20A and 20B. However, no treatment was performed as a control.

As shown in (a) and (b) of FIG. 20, it can be confirmed that there is no cytotoxicity even when the microvesicles according to the present invention are treated to an amount of 1 X 10 ⁹ particles.

Since the specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

delete delete delete delete As a pharmaceutical composition for the prevention or treatment of intestinal diseases comprising extracellular vesicles derived from Kefir grain as an active ingredient,
The kefir grain is any one selected from the group consisting of Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefirgranum,
The intestinal disease is irritable bowel syndrome or inflammatory bowel disease, pharmaceutical composition. delete The method of claim 5,
The microvesicles are derived from cultures of the kefir grains, pharmaceutical composition. delete The method of claim 5,
The inflammatory bowel disease is selected from the group consisting of Crohn's disease, bowel lesions accompanying Behcet's disease, ulcerative colitis, hemorrhagic rectal ulcer and ileal cystitis. delete delete delete delete delete delete As a food composition for the prevention or improvement of intestinal diseases, including extracellular vesicles derived from Kefir grain as an active ingredient,
The kefir grain is any one selected from the group consisting of Lactobacillus kefiranofaciens, Lactobacillus kefiri and Lactobacillus kefirgranum,
The bowel disease is irritable bowel syndrome or inflammatory bowel disease, food composition. delete

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